Bi-allelic variants in DOHH, catalyzing the last step of hypusine biosynthesis, are associated with a neurodevelopmental disorder.

Deoxyhypusine hydroxylase (DOHH) is the enzyme catalyzing the second step in the post-translational synthesis of hypusine [Nε-(4-amino-2-hydroxybutyl)lysine] in the eukaryotic initiation factor 5A (eIF5A). Hypusine is formed exclusively in eIF5A by two sequential enzymatic steps catalyzed by deoxyhypusine synthase (DHPS) and deoxyhypusine hydroxylase (DOHH). Hypusinated eIF5A is essential for translation and cell proliferation in eukaryotes, and all three genes encoding eIF5A, DHPS, and DOHH are highly conserved throughout eukaryotes. Pathogenic variants affecting either DHPS or EIF5A have been previously associated with neurodevelopmental disorders. Using trio exome sequencing, we identified rare bi-allelic pathogenic missense and truncating DOHH variants segregating with disease in five affected individuals from four unrelated families. The DOHH variants are associated with a neurodevelopmental phenotype that is similar to phenotypes caused by DHPS or EIF5A variants and includes global developmental delay, intellectual disability, facial dysmorphism, and microcephaly. A two-dimensional gel analyses revealed the accumulation of deoxyhypusine-containing eIF5A [eIF5A(Dhp)] and a reduction in the hypusinated eIF5A in fibroblasts derived from affected individuals, providing biochemical evidence for deficiency of DOHH activity in cells carrying the bi-allelic DOHH variants. Our data suggest that rare bi-allelic variants in DOHH result in reduced enzyme activity, limit the hypusination of eIF5A, and thereby lead to a neurodevelopmental disorder.

Neuron-specific ablation of eIF5A or deoxyhypusine synthase leads to impairments in growth, viability, neurodevelopment, and cognitive functions in mice.

Eukaryotic initiation factor 5A (eIF5A)†,‡ is an essential protein that requires a unique amino acid, hypusine, for its activity. Hypusine is formed exclusively in eIF5A post-translationally via two enzymes, deoxyhypusine synthase (DHPS) and deoxyhypusine hydroxylase. Each of the genes encoding these proteins, Eif5a, Dhps, and Dohh, is required for mouse embryonic development. Variants in EIF5A or DHPS were recently identified as the genetic basis underlying certain rare neurodevelopmental disorders in humans. To investigate the roles of eIF5A and DHPS in brain development, we generated four conditional KO mouse strains using the Emx1-Cre or Camk2a-Cre strains and examined the effects of temporal- and region-specific deletion of Eif5a or Dhps. The conditional deletion of Dhps or Eif5a by Emx1 promotor-driven Cre expression (E9.5, in the cortex and hippocampus) led to gross defects in forebrain development, reduced growth, and premature death. On the other hand, the conditional deletion of Dhps or Eif5a by Camk2a promoter-driven Cre expression (postnatal, mainly in the CA1 region of the hippocampus) did not lead to global developmental defects; rather, these KO animals exhibited severe impairment in spatial learning, contextual learning, and memory when subjected to the Morris water maze and a contextual learning test. In both models, the Dhps-KO mice displayed more severe impairment than their Eif5a-KO counterparts. The observed defects in the brain, global development, or cognitive functions most likely result from translation errors due to a deficiency in active, hypusinated eIF5A. Our study underscores the important roles of eIF5A and DHPS in neurodevelopment.

Elevation of cellular Mg2+ levels by the Mg2+ transporter, Alr1, supports growth of polyamine-deficient Saccharomyces cerevisiae cells.

The polyamines putrescine, spermidine, and spermine are required for normal eukaryotic cellular functions. However, the minimum requirement for polyamines varies widely, ranging from very high concentrations (mm) in mammalian cells to extremely low in the yeast Saccharomyces cerevisiae Yeast strains deficient in polyamine biosynthesis (spe1Δ, lacking ornithine decarboxylase, and spe2Δ, lacking SAM decarboxylase) require externally supplied polyamines, but supplementation with as little as 10-8 m spermidine restores their growth. Here, we report that culturing a spe1Δ mutant or a spe2Δ mutant in a standard polyamine-free minimal medium (SDC) leads to marked increases in cellular Mg2+ content. To determine which yeast Mg2+ transporter mediated this increase, we generated mutant strains with a deletion of SPE1 or SPE2 combined with a deletion of one of the three Mg2+ transporter genes, ALR1, ALR2, and MNR2, known to maintain cytosolic Mg2+ concentration. Neither Alr2 nor Mnr2 was required for increased Mg2+ accumulation, as all four double mutants (spe1Δ alr2Δ, spe2Δ alr2Δ, spe1Δ mnr2Δ, and spe2Δ mnr2Δ) exhibited significant Mg2+ accumulation upon polyamine depletion. In contrast, a spe2Δ alr1Δ double mutant cultured in SDC exhibited little increase in Mg2+ content and displayed severe growth defects compared with single mutants alr1Δ and spe2Δ under polyamine-deficient conditions. These findings indicate that Alr1 is required for the up-regulation of the Mg2+ content in polyamine-depleted cells and suggest that elevated Mg2+ can support growth of polyamine-deficient S. cerevisiae mutants. Up-regulation of cellular polyamine content in a Mg2+-deficient alr1Δ mutant provided further evidence for a cross-talk between Mg2+ and polyamine metabolism.

The Yeast Heterochromatin Protein Sir3 Experienced Functional Changes in the AAA+ Domain After Gene Duplication and Subfunctionalization.

A key unresolved issue in molecular evolution is how paralogs diverge after gene duplication. For multifunctional genes, duplication is often followed by subfunctionalization. Subsequently, new or optimized molecular properties may evolve once the protein is no longer constrained to achieve multiple functions. A potential example of this process is the evolution of the yeast heterochromatin protein Sir3, which arose by duplication from the conserved DNA replication protein Orc1 We previously found that Sir3 subfunctionalized after duplication. In this study, we investigated whether Sir3 evolved new or optimized properties after subfunctionalization . This possibility is supported by our observation that nonduplicated Orc1/Sir3 proteins from three species were unable to complement a sir3Δ mutation in Saccharomyces cerevisiae To identify regions of Sir3 that may have evolved new properties, we created chimeric proteins of ScSir3 and nonduplicated Orc1 from Kluyveromyces lactis We identified the AAA+ base subdomain of KlOrc1 as insufficient for heterochromatin formation in S. cerevisiae In Orc1, this subdomain is intimately associated with other ORC subunits, enabling ATP hydrolysis. In Sir3, this subdomain binds Sir4 and perhaps nucleosomes. Our data are inconsistent with the insufficiency of KlOrc1 resulting from its ATPase activity or an inability to bind ScSir4 Thus, once Sir3 was no longer constrained to assemble into the ORC complex, its heterochromatin-forming potential evolved through changes in the AAA+ base subdomain.